Cells apoptosis induced by intense heat stress is the prominent feature of heat-related illness. However, little is known about the biological effects of heat stress on cells apoptosis. Herein, we presented evidence that intense heat stress could induce early apoptosis of HUVEC cells through activating mitochondrial pathway with changes in mitochondrial membrane potential(ΔΨm), release of cytochrome c, and activation of caspase-9 and -3. We further revealed that p53 played a crucial role in heat stress-induced early apoptosis, with p53 protein rapidly translocated into mitochondria. Using pifithrin-α(PFT), a p53's mitochondrial translocation inhibitor, we found that pretreated with PFT, heat stress induced mitochondrial p53 translocation was significantly suppressed, accompanied by a significant alleviation in the loss of ΔΨm, cytochrome c release and caspase-9 activation. Furthermore, we also found that generation of reactive oxygen species (ROS) was a critical mediator in heat stress-induced apoptosis. In addition, the antioxidant MnTMPyP significantly decreased the heat stress-induced p53's mitochondrial translocation, followed by the loss of ΔΨm, cytochrome c release, caspase-9 activation and heat stress-mediated apoptosis. Conclusively, these findings indicate the contribution of the transcription-independent mitochondrial p53 pathway to early apoptosis in HUVEC cells induced by oxidative stress in response to intense heat stress.